Accumulation of copper in the kidney of pigs fed high dietary zinc is due to metallothionein expression with minor effects on genes involved in copper metabolism

A study was conducted to determine the effect of high dietary zinc (Zn) oxide on trace element accumulation in various organs with special emphasis on the kidney. A total of 40 weaned piglets were allocated into two groups with 16 and 24 piglets each receiving a diet containing normal (NZn; 100 mg Zn/kg) or high (HZn; 2,100 mg Zn/kg) Zn concentration, respectively. After two weeks, eight piglets from each treatment were killed and organ samples were taken. Eight piglets from the remaining 16 pigs fed HZn diets were changed to NZn diets (CZn). All remaining piglets were killed after another two weeks for organ sampling. Trace element concentration was determined in the jejunum, liver, kidney, pancreas, bone (metacarpal IV), spleen, lung, thymus, tonsils and lymph nodes of jejunum, ileum and colon. Kidney mRNA expression of Zn transporter ZnT1 and ZIP4, genes involved in Cu metabolism (Ctr1, Atox1, SOD1, ATP7A, CCS, CP) and divalent metal ion transport (DMT1) and binding (MT-1a, MT-2b, MT-3) were determined. The Zn concentration in jejunum, liver, pancreas tissue and metacarpal IV was higher (P < 0.05) in HZn group compared with NZn and CZn groups. Trace element concentration in organs of CZn pigs was similar to those fed NZn diets. Zn concentration in muscle, lung and lymphatic organs as thymus, tonsils, spleen and lymph nodes of jejunum, ileum and colon did not differ between the groups. Zn and Cu were positively correlated (R = 0.67; P < 0.05) in the kidney. No significant differences for Cu chaperones, Cu transporters and Cu-dependent factors were determined despite decreased expression of Atox1 after two weeks and increased Ctr1 expression over time in the HZn group. Expression of MT-1a, MT-2b and MT-3 were significantly higher in HZn fed pigs with most pronounced effects for MT-1a > MT-2b > MT-3. Gene expression of MTs in pigs fed CZn diets did not differ from pigs fed NZn diets. The data suggest that high dietary Zn feeding in pigs leads to Cu co-accumulation in the kidney of pigs with minor effect on genes relevant for Cu metabolism. In addition, the organ Zn and Cu accumulation is reversible after two weeks of withdrawal of high dietary Zn.     

Biochemical characteristics of phytases from fungi and the transformed microorganism

Five sources of phytases were used to study their biochemical characteristics. Phytase E was from an original Escherichia coli (E. coli), phytase PI and PG from the transformed Pichia pastoris (P. pastoris) with phytase gene of E. coli, phytase B and R from Aspergillus niger (A. niger). The results showed that the relative phytase activities had no significant changes when temperature was below 60 °C (P>0.05), and then decreased significantly with temperature increasing (P<0.01). The fungal phytase with the phytase gene from A. niger had the higher thermostability than the bacterial phytase with the phytase gene from E. coli; i.e. at 70 °C, 27–58% of phytase activity (compared with 30 °C) was retained for the bacterial phytase, and 73–96% for the fungal phytase; at 90 °C, 20–47% was retained for the bacterial phytase, and 41–52% for the fungal phytase, especially for the most thermostable phytase R (P<0.01). The optimum pH ranges were 3.0–4.5 for the bacterial phytases and 5.0–5.5 for the fungal phytases (P<0.01). When pH levels were 1, 7 and 8, only 3–7% of phytase activity (compared with the maximum phytase activity at a pH point) was retained for both bacterial and fungal phytases. The amount of inorganic P released from soybean meal was significantly increased when the levels of phytase activity in the soybean meal increased from 0 to 1.0 U/g soybean meal (P<0.01), except for phytase PI. The maximum P released was obtained at 1 U/g soybean meal for all five kinds of phytases (P<0.01). The most economical phytase concentration for P released was 0.25 U/g for phytase PI and B, and 0.50–1.0 U/g for phytase PG, E and R. In addition, the linear and non-linear regression models were established to estimate phytase activity and its characteristics very easily and economically.     

Influence of supplemental enzymes on the performance and phosphorus excretion of broilers fed wheat-based diets to 6 weeks of age

Efficacies of phosphorolytic enzymes (phytase+acid phosphatase), and an enzymic “cocktail” (phytase+acid phosphatase+pectinase+citric acid), were investigated in broilers fed wheat-based diets from day 1 to 43. Broilers were fed the following four diets: (1) a positive control diet (7.1 g total P/kg, 4.1 g non-phytate P/kg, 9.8 g Ca/kg); (2) a low phosphorus diet (4.1 g total P/kg, 1.7 g non-phytate P/kg, 6.0 g Ca/kg) supplemented with phytase (750 units/kg) and acid phosphatase (3156 units/kg); (3), a low phosphorus diet (4.1 g total P/kg, 1.7 g non-phytate P/kg, 6.0 g Ca/kg) supplemented with phytase, acid phosphatase, pectinase (1900 units/g) and citric acid (20 g/kg); and (4) a low phosphorus diet (4.1 g total P/kg, 1.7 g non-phytate P/kg, 8.0 g Ca/kg) supplemented as in diet 3. For the grower period (22–43 days), the contents of P and Ca were lowered by 0.2 and 0.3 g/kg, respectively. The dietary treatments were fed to three floor pen replicates of 50 birds each. For the starter period, there were no differences observed among dietary treatments in terms of body weight gains or feed efficiency. Total body weight gains for the starter and grower periods did not differ among dietary treatments, but total feed efficiency was significantly enhanced in birds fed diet 2. At the completion of the experiment chickens fed phosphorolytic enzymes had the best feed efficiencies, the highest contents of ash in the toes, and the highest carcass yield. Chicken receiving the cocktail of enzymes and 8 g Ca/kg (diet 4) performed as well as birds in the control treatment, but had higher yields of carcass and excreted 56% less phosphorus.     

Interaction of dietary zinc and growth implants on weight gain,carcass traits and zinc in tissues of growing beef steers and heifers

In two separate studies, 60 beef heifers (379 kg BW) and 60 beef steers (348 kg BW) were randomly assigned to six treatments in 2×3 factorial arrangements. The treatments were with or without Synovex^® implants combined with either a control diet or diets supplemented with 200 ppm Zn from ZnSO₄ or zinc methionine (Zn-Met). Near the mid-point of the feeding periods, cattle were vaccinated with a modified live virus and subsequent titers and concentrations of immunoglobulin G (IgG) were measured. Liver and blood samples were obtained 1 week prior to the start of the experiments and at intervals during the experiments. In experiment 1, average daily gains of beef heifers were (P<0.05) affected by the interaction of implant and source of dietary Zn. Compared to control and ZnSO₄ treatments, supplementation with Zn-Met increased (P<0.05) the concentration of Zn in serum. Antibody titers and concentrations of IgG in serum were highest (P<0.05) in heifers fed ZnSO₄ compared to heifers fed the control or Zn-Met supplemented diets. The Synovex-H^® implant reduced the concentrations of Zn and Cu in liver. In experiment 2, Synovex-S^® implants improved (P<0.05) weight gains of steers supplemented with 200 ppm dietary Zn from ZnSO₄ compared to non-implanted steers. However, the implant had no effect when Zn-Met was the dietary Zn source. The implant increased (P<0.05) concentrations of Zn in liver of steers supplemented with 200 ppm dietary Zn and reduced Zn in liver of steers fed the control diet. Implanted steers had higher (P<0.05) Cu status and IgG concentrations in serum than non-implanted steers. Steers supplemented with either ZnSO₄ or Zn-Met had greater (P<0.05) concentrations of Zn in liver and plasma than steers fed the control diet. These results indicate both the level and source of Zn supplementation in diets of feedlot cattle affect their response to growth implants.     

Effect of copper sources and levels on serum lipid profiles in Black Bengal (Capra hircus) kids

Twenty eight 2–3 month old castrated male Black Bengal kids (Capra hircus) were used to determine the effects of dietary Cu concentration on lipid metabolism. These kids were randomly assigned to one of seven treatments in a ((2 × 3) + 1) factorial arrangement. Factors were two sources of Cu (CuSO₄ versus Cu proteinate) fed at three dietary levels (10, 20 or 30 mg/kg) and the control group, where neither CuSO₄ nor Cu proteinate were supplemented. Kids were fed a basal diet containing maize (19.5%), soybean (17.0%), deoiled rice bran (56.5%), molasses (4.0%), di-calcium phosphate and salt (1.0% each), and mineral and vitamin mixture (0.5% each) supplements, at 3.5% of BW to meet NRC requirements for protein, energy, macro minerals and micro minerals, excluding Cu. The basal diet (DM basis) contained 5.7 mg Cu/kg, 122.5 mg Fe/kg, 110 mg Zn/kg, 0.26 mg Mo/kg and 0.32% S. CuSO₄ or Cu proteinate (Cu-P) was added to the basal diet at the rate of 10, 20 and 30 mg/kg. Kids were housed in a well-ventilated shed with facilities for individual feeding in aluminum plated metabolic cages in an open-sided barn. Blood samples were collected on Days 0, 30, 60 and 90 to determine serum cholesterol, high density lipoprotein (HDL), total lipid and phospholipids. Kids were slaughtered after metabolism trial and liver tissues were collected to determine the copper and zinc concentrations. Kids receiving Cu-P showed higher (P < 0.05) HDL, total lipid and phospholipid concentrations. Increase in dietary level of Cu significantly decreased (P < 0.05) serum cholesterol and increased serum HDL, total lipid and phospholipid concentrations. There was an increasing (P < 0.05) trend in liver Cu with the increased dietary level of Cu supplementation irrespective of source, but the increasing rate was greater with CuSO₄ than Cu-P supplementation. Kids’ diet containing 30 mg/kg CuSO₄ had 26% more liver Cu than those fed iso-amounts of Cu-P. Fecal Cu excretion was increased with the increasing dietary level of Cu, and excretion was reduced by the use of Cu-P in the diet. In conclusion, dietary supplementation of organic Cu in the form of copper proteinate had significant effects on lipid metabolism in goat kids. There was an increase in accumulation of Cu in the liver and excretion of Cu in feces with the increase of dietary level of Cu in the diet of Black Bengal kids.     

Copper and zinc in Elodea canadensis from rivers with various pollution levels

The anthropogenic impact of xenobiotics contributes to environmental risk for the aquatic environment and thus, must be controlled. Elodea canadensis, a cosmopolitan aquatic macrophyte with an important role in the ecology of many littoral zones, may provide an integrated record of pollution. Therefore, it was interesting to investigate the accumulation of Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn in this species and in water and bottom sediments collected from rivers with various levels of contamination. Of these rivers one control and one polluted was selected for the collection of E. canadensis for an experiment to compare the ability of this species to accumulate Cu and Zn. These elements were supplemented at concentrations (mg L⁻¹) of 0.01, 0.02, 0.03, 0.05, 0.08 and 0.14 as CuSO₄·5H₂O, and 0.4, 0.6, 0.9, 1.4, 2.03 and 3.04 as ZnSO₄·7H₂O and in a mixture containing (mg L⁻¹) 0.01Cu + 0.4Zn, 0.02Cu + 0.6Zn, 0.03Cu + 0.9Zn, 0.05Cu + 1.4Zn, 0.08Cu + 2.03Zn and 0.14Cu + 3.04Zn. After the experiment, E. canadensis from the polluted river contained significantly higher Cu and Zn concentrations when applied separately and also significantly higher Cu and Zn concentrations when applied as a mixture compared to the control river. These higher concentrations in E. canadensis from the polluted river were found in all combinations in the experiment. Thus, E. canadensis habituated in polluted sites to the exposure, and long-term influence of elevated metal levels appeared to be better adapted, and it also exhibited a higher increase in biomass than plants from the control river in all the experimental Cu and Zn solutions. Younger leaves of E. canadensis were more resistant to the effects of Cu and Zn than older leaves. Both Cu and Zn negatively affected the cell structure of older leaves, although the influence of Cu on plasma membrane integrity and chloroplast distribution was stronger than that of Zn. The influence of the Cu + Zn mixture on E. canadensis resulted in less pronounced cell disintegration than the influence of Cu added separately.The explanation of differences in the E. canadensis biomass increase and metal concentrations under the binary Cu and Zn impact needs further examination.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Estimation of the phosphorus requirement of weanling pigs fed supplemental phytase

Studies were conducted with crossbred weanling pigs to determine the level of phosphorus needed to be fed when a maize–soyabean meal–whey diet was supplemented with exogenous phytase (Natuphos™). In Trial 1, phytase was added at 1200 phytase units (PTU) kg⁻¹ as phosphorus decreased. The control diet in Phase I (0–14 days) contained 7.3 g kg⁻¹ phosphorus and in Phase II (14–28 days) contained 6.5 g kg⁻¹ phosphorus. Dietary phosphorus was calculated to decrease by 0.8, 1.6 or 2.4 g kg⁻¹ when phytase was supplemented. Chromic oxide was added for estimation of apparent absorption of phosphorus. Performance was optimum when 5.7 and 4.8 g kg⁻¹ phosphorus (analysed levels) were fed with 1200 PTU kg⁻¹ phytase in Phases I and II, respectively. The lowest dietary phosphorus levels did not reduce performance for the overall 28-day period. Apparent phosphorus digestibility was increased by phytase in Phase I when 5.7 g kg⁻¹ phosphorus was fed compared to the control diet and in Phase II when 6.0 g kg⁻¹ phosphorus was fed with phytase. Faecal phosphorus excretion decreased in both phases as dietary phosphorus decreased. Faecal phosphorus excretion was minimized at the lowest phosphorus level with no decrease in performance. The estimated requirement for dietary phosphorus, as determined by the NLIN procedure, is 5.0 g kg⁻¹ in Phase I and 4.3 g kg⁻¹ in Phase II when 1200 PTU kg⁻¹ is used. In Trial 2, phytase was supplemented at 500 PTU kg⁻¹ when phosphorus was decreased in the diet. The control diet contained 6.6 and 6.0 g kg⁻¹ phosphorus in Phases I and II, respectively, and phosphorus was calculated to decrease by 0.5, 1.0, 1.5, or 2.0 g kg⁻¹ when phytase was added. Daily gain decreased when 5.0 g kg⁻¹ phosphorus was fed in Phase I and when 4.6 or 4.2 g kg⁻¹ (analysed levels) phosphorus was fed in Phase II with 500 PTU kg⁻¹. Faecal phosphorus excretion decreased as dietary phosphorus decreased, but there were no treatment effects on apparent phosphorus digestibility. The dietary phosphorus requirement was estimated to be 5.7 and 5.0 g kg⁻¹ in Phases I and II, respectively, when phytase is fed at 500 PTU kg⁻¹. At the present recommendation of 500 PTU kg⁻¹ in starter feed, phosphorus can be decreased by 0.10 g kg⁻¹. However, higher levels of phytase are needed to actually increase apparent phosphorus digestibility.     

Ruminal ochratoxin A degradation—Contribution of the different microbial populations and influence of diet

The mycotoxin ochratoxin A (OTA) is degraded extensively in the rumen. In this study, the relative contribution of different rumen microbial populations (MP) and the effect of diet on degradation of OTA were evaluated in a factorial design experiment. Degradation of OTA was quantified by using the Hohenheim gas test (HGT) in vitro fermentation system. Five different HGT diets were used (concentrate:forage proportions (C:F) – 10:90, 30:70, 50:50, 70:30, 90:10), and donor animals were fed diets with the respective ratio. Diets with the highest concentrate content were supplied with and without 10 g/kg sodium bicarbonate (70:30 BC and 90:10 BC). The MP included whole rumen fluid, fungi + protozoa, bacteria + protozoa, protozoa and bacteria + fungi. Protozoa numbers were counted after 24 h and OTA and ochratoxin alpha (OTα) analysed at 0, 4, 8, 12, 24 h. Area under the curve (AUC) and half-life were calculated for the latter two. The short average OTA half-life for whole rumen fluid of 2.6 h (1.3–4.5 h) demonstrates the high OTA degradation capacity of the rumen MP (i.e., standard HGT inoculum) and corresponds well with published in vivo results. Both MP and diet affected OTA degradation. Interactions among factors occurred (P<0.001), which made it necessary to do further comparisons within factor levels. Among MP, those with bacteria (bacteria + fungi and bacteria + protozoa) had lower AUC values (P<0.001) for OTA (196–673 ng/ml h, meaning higher degradation capacity, than those without bacteria (fungi + protozoa and protozoa; 701–1206 ng/ml h). Whole rumen fluid had the lowest AUC values (146–249 ng/ml h; P<0.05). Diet had a quadratic effect (P=0.001) on protozoal numbers with minimum values for the lowest and highest C:F ratios, for bacteria + protozoa, fungi + protozoa and protozoa, but no corresponding effect was found for OTA degradation parameters. While the generally high capacity to degrade OTA was confirmed, results for the contribution of different microbial groups shed new light on ruminal OTA degradation.     

Trace mineral status and liver and blood parameters in sheep without mineral supply compared to local roe deer (Capreolus capreolus) populations

General health, clinical-chemical blood analysis and copper (Cu), zinc (Zn), selenium (Se) and vitamin E concentrations in plasma and liver tissue (wet weight, ww) of two extensive grazing sheep flocks without mineral supply were compared to the status of local roe deer (Capreolus capreolus) populations (liver samples). Both sheep flocks were classified as healthy except for a remarkable variation in body weight and a slight foot rot infection in one flock. Hematology of sheep was normal, and total protein and creatinine as well as activities of creatin kinase, aspartat-amino-transferase, alkaline phosphatase and gamma-glutamyl-transferase in plasma were within reference levels. The mean of glutamate dehydrogenase (13.8 U/l) was slightly elevated in one flock. Mean liver concentrations of Zn (38.9 and 43.5 mg/kg ww) and Cu (111 and 87.5 mg/kg ww) in sheep flocks were higher compared to the respective roe deer populations (27.5 and 36.3 mg Zn/kg ww; 18.3 and 28.6 mg Cu/kg ww). This is supposed to be caused by differences in Cu and Zn metabolism in sheep and roe deer. Selenium deficiency was diagnosed in liver samples of both sheep flocks (0.21 and 0.23 mg/kg ww). There were neither significant differences compared to roe deer (0.21 and 0.27 mg Se/kg ww) nor differences depending on location. Correlations between plasma and liver concentrations of Cu, Zn and Se were not significant in sheep. Means of vitamin E in liver samples (30.6 and 41.8 mg/kg ww) were higher in roe deer populations. This may be caused by the opportunity of selective browsing for wild ruminants, which allows access to younger plants which are higher in vitamin E.     

Influence of diet complexity on productive performance and nutrient digestibility of weanling pigs

The effect of diet complexity on coefficient of total tract apparent digestibility (CTTAD) and growth performance was studied in piglets from 21 to 62 d of age. There were five experimental prestarter diets (21–41 d of age) with similar net energy and total indispensable amino acids content. The negative control diet contained 400 g raw maize, 40 g fish meal (FM) and 70 g lactose (LAC)/kg and the positive control diet contained 400 g cooked maize, 100 g FM and 140 g LAC/kg. The other three diets were similar to the positive control diet but the cooked maize was substituted by raw maize or contained 40 g FM/kg or 70 g LAC/kg, respectively. Each treatment was replicated six times (six pigs per pen). For the starter period (42–62 d of age), half of the pens of each of the prestarter treatments was sorted into two groups and fed either a standard soybean meal–raw maize–lard diet or a diet with similar nutrient profile that included 200 g cooked maize, 50 g FM, 13 g LAC, 20 g soy protein concentrate and 10 g soybean oil/kg in substitution of lower cost ingredients. Dietary treatment did not affect piglet performance at any age, but incidence of diarrhoea during the prestarter period, was higher in piglets fed the negative control diet than in piglets fed any of the other diets (P<0.05). At 30 d of age (prestarter period), the CTTAD of organic matter and gross energy were lower (P<0.001) for pigs fed the negative control diet than for pigs fed the other diets, but that of crude protein was not affected. At 50 d of age (starter period), dietary treatment did not affect the CTTAD of any dietary component. It is concluded that the use of high levels of high quality ingredients in the diet did not improve growth performance of piglets at any age. From 21 to 41 d of age, the incidence of diarrhoea was reduced and the CTTAD of dietary components was increased when the more complex diets were fed. The inclusion of high levels of high quality ingredients in the diet to maximize performance of young pigs might not be justified under all circumstances.     

New insights into the chemical and isotopic composition of human-body biominerals. I: Cholesterol gallstones from England and Greece

We have analyzed gallstones from four patients of Europe and particularly from England (including samples from a mother and a daughter) and Greece. According to the XRD, FTIR, NMR and laser micro-Raman results the studied materials correspond to typical cholesterol monohydrate (ChM). The micro-morphology of cholesterol microcrystals was investigated by means of SEM–EDS. The XRF results revealed that Ca is the dominant non-organic metal in all gallstones (up to ～1.95 wt.%) together with Fe, Cu, Pb and Ni (up to ～19 ppm for each metal). Gallstones from England contain additional Mn (up to ～87 ppm) and Zn (up to ～6 ppm) while the sample of the mother contains negligible Zn and Mn, compared to that of her daughter, but significant As (～4.5 ppm). All cholesterol gallstones examined are well enriched in potentially toxic metals (Pb, as well as Ni in one case) and metalloids (As also in one case) as compared to the global average. The position of Zn, which is a characteristic biometal, in the structure of cholesterol, was investigated by molecular simulation using the Accelrys Materials Studio^® software. On the basis of IRMS results, all gallstones examined exhibit a very light δ¹³C signature (average δ¹³C ～?24‰ PDB). Gamma-ray spectrometry measurements indicate the presence of ²¹⁴Pb and ²¹⁴Bi natural radionuclides due to the ²³⁸U series as well as an additional amount of ⁴⁰K.     

l-Lysine supplementation does not affect the bioavailability of copper or iron in rats

l-lysine (Lys) is an essential amino acid that is added to foods and dietary supplements. Lys may interact with mineral nutrients and affect their metabolism. This study examined the effect of dietary Lys supplementation on the bioavailability of copper (Cu) and iron (Fe). Weanling male Sprague-Dawley rats were fed one of five diets (20% casein) for 4 weeks containing normal Cu and Fe (control) or low Cu or Fe without (LCu, LFe) or with (LCu + Lys, LFe + Lys) addition of 1.5% Lys. Final body weights, body weight gains and food consumption of the rats did not differ (P ≥ 0.05) among diet groups. Rats fed the low Cu or Fe diets showed changes in nutritional biomarkers compared to control rats, demonstrating reduced Cu and Fe status, respectively. Hematological parameters, serum ceruloplasmin activity and Cu and Fe concentrations in serum, liver, kidney and intestinal mucosa were unaffected (P ≥ 0.05) by Lys supplementation. These results indicate that in the context of an adequate protein diet, Lys supplementation at a relatively high level does not affect Cu or Fe bioavailability in rats.     

Metal complexes of crosslinked chitosans: Correlations between metal ion complexation values and thermal properties

A series of heavy metal complexes of crosslinked chitosans were evaluated by thermogravimetric studies. The metal complexes with Cu, Cd and Hg ions exhibiting the highest complexing ability to chitosans (Hg 354–364, Cu 100–112, and Cd 121–160, in mg/g chitosan), had the lowest onset of degradation temperatures (range 194–210 °C) and the lowest final degradation temperatures (generally less than 294–304 °C for Hg, 296–338 °C for Cu, and 305–368 °C for Cd complexes). Mn ion, with the lowest binding to chitosans (Mn 5–7 mg/g), showed the reverse behavior, having onset (240–248 °C) and final degradation temperatures (range 300–368 °C). Zn (binding 74–87 mg/g) and Pb (binding 39–62 mg/g) ions have a binding ability intermediate to Cu/Cd/Hg and Mn extremes, and therefore the effects on onset and final degradation temperatures are intermediate to these values.     

EDTA disodium zinc has superior bioavailability compared to common inorganic or chelated zinc compounds in rats fed a high phytic acid diet

Different zinc (Zn) compounds have unique properties that may influence the amount of Zn absorbed particularly in the presence of phytic acid (PA), a common food component that binds Zn and decreases its bioavailability. In this study, 30-day-old male rats (n = 12/diet group) were fed diets supplemented with PA (0.8%) and low levels (8 mg Zn/kg diet) of inorganic (Zn oxide, Zn sulphate) or chelated (Zn gluconate, Zn acetate, Zn citrate, EDTA disodium Zn, Zn orotate) Zn compounds for 5 weeks. Two control groups were fed diets supplemented with low or normal (30 mg Zn/kg diet) Zn (as Zn oxide) without added PA. Control rats fed the low Zn oxide diet showed depressed Zn status. Addition of PA to this diet exacerbated the Zn deficiency in rats. Growth (body weight gain and femur length) and Zn concentrations in plasma and tissues were similar in rats fed Zn oxide, Zn sulphate, Zn gluconate, Zn acetate, Zn citrate or Zn orotate. Rats fed EDTA disodium Zn showed enhanced growth compared to rats fed Zn oxide or Zn gluconate and had higher Zn concentrations in plasma and femur compared to rats fed all other Zn compounds. Only the haematological profile of rats fed EDTA disodium Zn did not differ from control rats fed normal Zn. These data indicate that in rats fed a high PA diet, bioavailability of commonly used inorganic or chelated Zn compounds does not differ appreciably, but Zn supplied as an EDTA disodium salt has superior bioavailability.     

Extracellular expression of a thermostable phytase (phyA) in Kluyveromyces lactis

Functional expression of a thermostable phytase from A. niger was achieved in Kluyveromyces lactis GG799 cells. Effective secretion of recombinant enzyme (198 U ml⁻¹) in the fermentation broth at 72 h incubation at 22 °C was obtained. Purified enzyme showed a specific activity of 72 U mg⁻¹) and was detected on SDS-PAGE as a heavily glycosylated protein with a molecular weight of ≥140 kDa. Optimum temperature of the enzyme was at 55 °C and it showed a characteristic bi-hump pH profile with two pH optima (at pH 2.5 and 5.5). Enzyme showed considerable pepsin resistance with 60% activity retention after incubation with pepsin at the ratio of 1:1000. Enzyme was thermostable retaining 69 and 37% activity at 90 and 100 °C for 10 min respectively and remained active at these temperatures till 1 h. Deglycosylation studies demonstrated negligible effect of N-linked glycans on thermal properties. Multiple sequence alignment data revealed a conserved Asn at position 345 of this phytase which might contribute to its thermal properties. This thermostable phytase coupled with its noticeable protease resistance could be a better alternative to current commercial phytases.     

Effects of zinc deficiency and zinc supplementation on homocysteine levels and related enzyme expression in rats

Methionine synthase (MS) and betaine-homocysteine methyltransferase (BHMT) are both zinc (Zn)-dependent methyltransferases and involved in the methylation of homocysteine. The objective of this study was to investigate the effects of dietary Zn supply on homocysteine levels and expression of the two enzymes in growing rats. Male weanling Sprague-Dawley rats were assigned randomly to four dietary groups (n = 8/group) for 3 weeks: Zn deficient (ZD; <1 mg Zn/kg); Zn control (ZC; 30 mg Zn/kg); Zn supplemented (ZS; 300 mg Zn/kg); pair fed (PF; 30 mg Zn/kg) to the ZD group. Serum and femur Zn concentrations were 83% and 58% lower in ZD, and 49% and 62% higher in ZS compared to ZC (P < 0.001), respectively. The ZD rats had lower feed intake (37%), body weight gains (45%), liver (43%) and kidney (31%) weights than those of ZC (P < 0.001), but these parameters in ZD were not significantly different from the PF controls. Serum homocysteine concentrations were 65% higher in ZD compared to PF (P < 0.05), and there was no significant difference in serum folate levels between ZD and PF groups. The mRNA expression of liver and kidney MS was 57% and 38% lower in ZD than PF (P < 0.001), respectively. Hepatic and renal BHMT mRNA levels were not altered in ZD compared to controls. The aforementioned measurements were not significantly different between ZS and ZC groups, except Zn levels. These results demonstrated that homocysteine homeostasis appeared to be disturbed by Zn deficiency but not Zn supplementation, and elevated serum homocysteine might be due to reduced expression of MS during Zn deficiency.     

Trace elements (copper,zinc, selenium and molybdenum) as markers in oral sub mucous fibrosis and oral squamous cell carcinoma

Oral cancer is a major cause of cancer morbidity and mortality worldwide and is prevalent in most areas where tobacco related practices are observed. Essential elements play a role in many biochemical reactions as a micro-source and there is growing evidence that their concentrations are altered on the onset and progress of malignant disease. In this study the levels of copper (Cu), zinc (Zn), selenium (Se) and molybdenum (Mo) in serum of patients with oral sub mucous fibrosis (OSMF) (n = 30) and oral squamous cell carcinoma (OSCC) (n = 30); were determined and the alterations of these critical parameters were analyzed in comparison with controls (n = 30) to identify predictors amongst these parameters for disease occurrence and progression. The serum Cu and Zn were established using Flame Atomic Absorption Spectrometry. Serum estimation of Se and Mo was done by graphite furnace atomic absorption spectrometry (GFAAS). Data analysis revealed a marked, progressive and significant increase in Cu levels in precancer (OSMF) and cancer (OSCC) groups as compared to the normal group. The level of Zn in serum was slightly elevated in OSMF and OSCC though not statistically significant. Cu/Zn ratio was slightly but not significantly elevated. Serum levels of Se and Mo were significantly decreased in the precancer and cancer groups as compared to the normals.     

Response of native grasses and Cicer arietinum to soil polluted with mining wastes: Implications for the management of land adjacent to mine sites

Mine tailings are an environmental problem in Southern Spain because wind and water erosion of bare surfaces results in the dispersal of toxic metals over nearby urban or agricultural areas. Revegetation with tolerant native species may reduce this risk. We grew two grasses, Lygeum spartum and Piptatherum miliaceum, and the crop species Cicer arietinum (chickpea) under controlled conditions in pots containing a mine tailings mixed into non-polluted soil to give treatments of 0%, 25%, 50%, 75% and 100% mine tailings. We tested a neutral (pH 7.4) mine tailings which contained high concentrations of Cd, Cu, Pb and Zn. Water-extractable metal concentrations increased in proportion to the amount of tailings added. The biomass of the two grasses decreased in proportion to the rate of neutral mine-tailing addition, while the biomass of C. arietinum only decreased in relation to the control treatment. Neutron radiography revealed that root development of C. arietinum was perturbed in soil amended with the neutral tailings compared to those of the control treatment, despite a lack of toxicity symptoms in the shoots. In all treatments and for all metals, the plants accumulated higher concentrations in the roots than in shoots. The highest concentrations occurred in the roots of P. miliaceum (2500 mg kg^?1 Pb, 146 mg kg^?1 Cd, 185 mg kg^?1 Cu, 2700 mg kg^?1 Zn). C. arietinum seeds had normal concentrations of Zn (70–90 mg kg^?1) and Cu (6–9 mg kg^?1). However, the Cd concentration in this species was ～1 mg kg^?1 in the seeds and 14.5 mg kg^?1 in shoots. Consumption of these plant species by cattle and wild fauna may present a risk of toxic metals entering the food chain.     

Subcellular fractionation of Cu exposed oysters,Crassostrea virginica,and Cu accumulation from a biologically incorporated Cu rich oyster diet in Fundulus heteroclitus in fresh and sea water

In order to examine the effect of salinity on Cu accumulation from a naturally incorporated diet, oysters (Crassostrea virginica) were exposed in sea water for 96 days to four waterborne [Cu]: 2.9 ± 0.7 (control), 4.3 ± 0.6, 5.4 ± 0.5, and 10.7 ± 1.0 µg L^? 1. After 96 days, the control whole body [Cu] increased from 2.1 ± 0.5 to 9.1 ± 1.1 µg g^? 1 w.w. and the highest [Cu] was 163.4 ± 27.1 µg g^? 1 w.w. in the oysters. Despite large differences in tissue [Cu], there was no effect on the fraction of trophically available metal in the oyster suggesting that trophic transfer will correlate well with tissue [Cu]. The control and highest [Cu] oysters became diet for killifish (Fundulus heteroclitus) in fresh and seawater for 40 days. The two diets contained 84.7 ± 5.1 and 850.5 ± 8.8 µg Cu g^? 1 d.w. Fish were fed a combined diet of oyster and a pellet supplement (20.5 ± 1.0 µg Cu g^? 1 d.w.) both at 5% body mass day^? 1. In killifish, Cu increased ~ 7% in gills and 100% in intestines after 6 weeks of exposure to the high Cu diet. No other tissues accumulated Cu above control levels. An 11-fold difference free Cu²⁺ concentrations was predicted in intestinal fluid between fresh and sea water, but there was no corresponding effect of salinity on intestinal Cu accumulation suggesting that Cu is not accumulated as the free ion.